Cancer, in its various forms, is one of the leading causes of death in developed countries and the failure to effectively treat many patients affected by these diseases drives the continued search for new treatment strategies. Angiogenesis is a physiological process in which new blood vessels are grown from pre-existing vessels. It is a vital process in normal growth and development but also plays a key role in pathological conditions, such as cancer by providing the vasculature needed to supply the growing tumour with oxygen and nutrients. Angiogenesis is regulated by the complex interplay between many pathways, such as those controlled by VEGF and Notch. The clinical relevance of angiogenesis has firmly established this process as a rational target for cancer therapy [1, 2]
Targeting tumour angiogenesis with anti-VEGF antibodies has been a successful strategy, with bevacizumab becoming licensed for several tumour types, either as single agent or in combination therapy [3]. However many patients do not respond, or their response is temporary [1]. There has also been efforts to target other components of the VEGF pathway, including antibodies to VEGFR1, VEGFR2, neuropilin receptors and PLGF [4]. It is of note that the toxicity of the antibodies is far less than that of small molecule inhibitors targeting VEGFR, implying much greater specificity and fewer off target effects.
The Notch pathway has been implicated in vascular homeostasis and patterning and in pathological angiogenesis, prompting research into its modulation for a therapeutic benefit [5]. Notch signalling is mediated by membrane-bound receptors (Notch1-4) and ligands (DII1, 3 and 4 and Jagged 1 and 2). Indeed, Notch1, Notch3, Notch4, DII1, DII4 and Jagged1 are expressed in cells of the vasculature and Notch1 [6], DII1 [7] and Jagged 1 [8] knockout mice are embryonic lethal due to vascular defects, whilst DII4 is haploinsufficient [9], thus suggesting that Notch signalling needs to be finely tuned to generate a fully functional vasculature. Indeed Notch pathway mutations are associated with human diseases exhibiting vascular defects, with NOTCH3 mutation in CADASIL [10] and JAGGED1 mutation in Alagille syndrome [11].
The binding of a Notch ligand, through its Delta/Serrate/Lag2 (DSL) domain, to the EGF11-12 region of a Notch receptor triggers proteolytic cleavage of the receptor and release of its intracellular domain (NICD). This processing requires the activity of two proteases, namely tumour necrosis factor α-converting enzyme (TACE) and presenilin/γ-secretase (a large protease complex made of presenilin1 or 2 as well as nicastrin, Pen-2 and Aph-1). The subsequent nuclear translocation of NICD results in transcriptional activation of genes of the Hes/E(spl) and Hey/Hesr families via the interaction of NICD with a member of the CSL (CBF1, Suppressor of hairless, and Lag-1) family of transcription factors also known as the recombination signal sequence-binding protein (RBP-Jk) and a transcriptional activator Mastermind-like protein (MAML). In turn, the Hes and Hey proteins, which are transcriptional repressors, inhibit the expression of genes that drive cells to adopt a differentiated fate [12].
In additional to playing a critical role in angiogenesis the Notch pathway is also implicated as an oncogenic pathway in tumour cells where it helps maintain stem cell populations, promotes cell survival, inhibits apoptosis and can drive cell proliferation (reviewed in [13]). Activating mutations in NOTCH1 have been identified in approximately 56% of T-cell acute lymphoblastic leukaemias (T-ALL) [14]; where they primarily act to induce either ligand-independent activation of the receptor or mutate the PEST domain and thus increase the stability of the Notch1 intracellular domain (NICD). Activating NOTCH1 mutations have also been identified in B-cell chronic lymphocytic leukaemia (B-CLL) [15, 16] and in mantle cell lymphoma [17] and these abnormalities have correlated with poor prognosis, suggesting that they define a distinct clinical subtype for therapeutic intervention. NOTCH2 mutations have been detected in 8% diffuse large B-cell lymphomas (DLBCL) and functionally demonstrated to be gain-of-function mutations [18]. Abnormal Notch signalling without the evidence of genetic lesions has also been reported in solid tumours, including breast, renal, pancreatic, prostate, cervical, endometrial, brain, intestinal, lung and skin cancers [19]. The pleiotrophic functions of Notch mean that this pathway can also have tumour suppressor roles in certain contexts (reviewed by [20, 21]). However, in the large majority of cases Notch signalling promotes tumour growth.
Thus Notch inhibition remains a promising new approach to cancer therapy and appears likely to be particularly useful in combination with other agents, including radiotherapy and chemotherapy [13]. Early studies of pan-Notch inhibition with γ-secretase inhibitors exhibited gastrointestinal toxicity; while prolonged treatment with anti-DII4 antibodies led to the development of vascular/endothelial cell-based tumours similar to hemangioblastoma (reviewed by [13]). More recently antibodies that functionally inhibit individual Notch receptors have shown anti-tumour effects without gut toxicity [22, 23]. Further analysis using inducible gut specific gene targeting to study the role of individual Notch ligands demonstrated that DII1 and DII4-mediated Notch signalling was required for the homeostasis of intestinal stem cells, whereas deletion of Jagged 1 did not perturb the intestinal epithelium [24]. Thus targeting Jagged 1 activated signalling via multiple Notch receptors should be feasible without adverse effects on gastrointestinal toxicity.
DII4 is highly expressed in the tumour vasculature and it influences tumour growth by regulating tumour angiogenesis in xenograft models [25-28] producing fewer but more functional vessels. Blocking DII4 signalling, either by expression of soluble DII4 or anti-DII4 antibodies, decreased tumour growth even in tumours resistant to VEGF inhibition [25] confirming this pathway as a therapeutic target. Interestingly, DII4 signalling induces expression of Jagged 1 [29]. This suggests that this latter Notch ligand may be a crucial downstream effector of DII4-Notch signalling in tumour angiogenesis. In addition, excessive Notch signalling due to high expression levels of Jagged 1 and Notch receptors has been described in several cancers. For example, overexpression of Jagged 1 and Notch1 in breast cancer is associated with poor overall survival with a synergistic effect of high-level co-expression [30]. In addition, expression of Jagged 1 in squamous cell carcinoma cells induces angiogenesis and of the four receptors Notch1 is known to be critical for adult angiogenesis [31, 32]. Interestingly, Jagged 1-triggered Notch signalling from cancer cells to the endothelium was shown to induce angiogenesis, generating vessels which were smaller than those induced by DII4.
Most blood vessels in the adult organism consist of at least two cell types, the endothelial cells (EC) and the mural cells (pericytes or the vascular smooth muscle cells (VSMC)). Pericytes wrap around the endothelium and play a crucial role in the stabilization and hemodynamic functions of the resulting blood vessels. In recent years, both cell types, EC and pericytes, have been targeted efficiently with the aim of oxygen starving solid tumours. Interestingly Notch signalling was shown to be crucial for the development of both these cell types [5].
Pericyte and vascular smooth muscle cell (VSMC) recruitment to newly forming vessels is a crucial step for vessel maturation and for endothelial cells to return to quiescence. A decade ago, pericytes were shown to stabilize immature blood vessels ending the plasticity period of vessel remodelling [33]. In addition, high vessel coverage by pericytes is critical to stabilise tumour vessels. Immature or poorly covered tumour vessels are extremely dependent on VEGF-A/VEGFR2 signalling and are therefore vulnerable to anti-VEGF therapies such as Bevacizumab. To date, however, the mechanisms underlying the survival of the remaining vasculature following anti-VEGF therapies are not fully understood. The remaining vessels appear to have greater pericyte coverage, suggesting that this cell type may limit the efficacy of these anti-angiogenic therapies [34, 35]. Interestingly, EC specific deletion of Jagged 1, which was recently shown to induce embryonic lethality and cardiovascular defects, was not due to impaired Notch1 signalling in EC or during arterial-venous differentiation. Instead deletion of Jagged 1 expression in EC was associated with poor vessel coverage by VSMC, which subsequently resulted in the developmental arrest of the mutant embryos [36]. Thus, based on its function as a regulator of vessel coverage, the Jagged 1/Notch signalling pathway presents itself as an attractive target that should have strong therapeutic benefit for the treatment of solid tumours, particularly when used in combination with anti-VEGF therapies. Therefore, antibodies aimed at blocking the Jagged 1-Notch signalling could have clinical benefit by targeting the tumour at three different levels, namely endothelial cells, mural cells and tumour cells.
Regulatory T cells (Tregs) infiltrate tumours in a vast array of tumour types. Their numbers are often clinically relevant and Tregs have a diversity of roles including their ability to suppress anti-tumour immunity and promote angiogenesis (recently reviewed by [80, 81]). Overexpression of J1 by antigen-presenting cells can induce human antigen-specific Tregs and modify the immune response to viral antigens [82]. Notch ligands, such as J1, are also expressed on Tregs and blockade of Notch signalling, using antibodies targeting J1 or N1 inhibited Treg suppressor function [83]. Furthermore, pre-exposure of CD4+CD25− effector T cells to J1 significantly increased their sensitivity to Treg-mediated suppression [84]. Thus, targeting J1 on immunoregulatory cell populations in the tumour microenvironment also offers the opportunity to improve the host immune response to tumour antigens.
There is new evidence suggesting that Jagged 1 may also be a ligand for a receptor other than the Notch family. CD46 (MCP) is a ubiquitously expressed human type I transmembrane glycoprotein that was originally discovered as a complement regulatory protein and then a cell-entry receptor enabling viral infection. Interestingly viral targeting of tumours using their elevated CD46 expression as a tumour selective entry receptor has been used as a strategy to facilitate therapy and imaging in multiple cancer types [37]. Anti-cancer applications include direct targeting of cancer cells by virally induced cytopathic effects and syncytial formation [38], including breast cancer [39], medulloblastoma [40], glioma [41] and recurrent ovarian cancer [42]; gene therapy targeting in lung adenocarcinomas [43]; and radiovirotherapy of prostate cancer [44]. More recently an immunomodulatory role in the co-stimulation of interferon-γ secreting effector human T helper type 1 (TH1) cells and their subsequent switch into IL-10-producing regulatory T cells has been identified (reviewed in [45].
There is a considerable literature regarding the complement system and cancer as reviewed recently [46, 47]. Most tumours express either soluble regulators or membrane bound complement receptors (CD35, CD46, CD55 and CD59) on the cell surface, thus suppressing activation of the complement system and diminishing its role in tumour clearance. It is well established that overexpression of CD46, CD55 and CD59 on tumours protects them from direct complement lysis. Furthermore, therapeutic antibodies (such as rituximab) use complement dependent cytotoxicity (CDC) to kill tumour cells and this activity can be increased by targeting membrane bound complement receptors e.g. using blocking antibodies.
CD46 upregulation in tumours has been widely reported and examples include upregulated expression in 77% of bladder cancers [48] and high expression in head and neck squamous cell carcinoma [49]. CD46 expression was more abundant on primary multiple myeloma cells than normal hematopoietic cells of various lineages in the bone marrow [50]. RNAi mediated knockdown of CD46 significantly inhibited the growth of pancreatic cancer cells overexpressing CD46 [51]. While breast cancers with CD46 expression have a less favourable prognosis [52] as did CD46+ ovarian cancer patients [53]. Downregulation of CD46 in MCF7 and MDA-MB-231 breast cancer cell lines via microRNAs induced opsonization of cancer cells via an alternative pathway resulting in complement activation [54]. RNAi targeting of CD46 in Du145 (prostate), BT474 (breast) and K562 (erythroleukaemia) cells also significantly increased C3 opsonization[55]. While shRNA targeting of CD46 and DAF enhanced complement mediated lysis in cervical cancer cells [56] and anti-sense phophorothioate oligonucleotides to down regulate CD55 and CD46 sensitized tumour cells to complement attack [57].
Some biological activities of CD46 cannot be explained by its interaction with the known ligands, for example C3b, and thus there has been speculation that another ligand existed for this receptor. In a recent study, activation of CD46 on CD4+ T cells was shown to regulate the expression of Notch and its ligands and furthermore Jagged 1 was identified as an additional physiological ligand for CD46 [45]. Further evidence of the importance of this interaction was that patients with mutations in genes encoding CD46 or Jagged 1 shared key biological features, including recurrent infections. While T-cell proliferation and effector function of TH2 cells was unaffected in these patients, the in vitro induction (or regulation) of TH1 cells was seriously compromised or absent and seemed to involve altered responsiveness to cytokines of the IL-2 family. The most significant cell surface receptor phenotype was deregulation of components of the IL-7 receptor, CD127 and CD132, which is required for T-cell homeostasis and enhancement of TH1 and TH17 responses. CD127 is also known to be a strong risk locus for multiple sclerosis, independently of the major histocompatibility complex.
The Jagged 1 binding site was localised to the CCP1 and CCP2 domains of CD46, these are the domains commonly bound by viral ligands such as adenovirus knob proteins or measles virus hemagglutinin. This Jagged 1 binding was mediated by a recombinant Jagged 1 DSL-EGF3 fragment comprising the DSL domain and the first three EGF-like domains demonstrating that the CD46− and Notch 1 binding sites in Jagged 1 are localised in the same region of the protein. Surface plasmon resonance experiments to measure the binding affinity of Jagged 1 interactions suggested that CD46 exhibited a tighter interaction with Jagged 1 than a soluble Notch 1 (EGF11-13) fusion protein. This was consistent with data suggesting that the presence of CD46 on T-cell surfaces restricts the interactions of Notch 1 and Jagged 1. Thus there is important cross-talk between the complement and Notch systems that is required for effector T-cell function and which may have a key role in other biological processes, particularly cancer.
Anti-Jagged 1 antibodies targeting the DSL domain and/or first three EGF repeats may thus also block the interaction between CD46 and Jagged 1. While some therapeutic effects may be mediated via the Notch system these reagents may also target additional pathways involving CD46.
The extracellular domain of Jagged 1 possesses shared structural features with all Notch ligands, these being the presence of a Delta/Serrate/Lag-2 (DSL) domain and a variable number of epidermal growth factor-like (EGF) domains. Crystallographic studies of the functional fragment of human Jagged 1 (DSL-EGF1-3) in combination with structure-informed mutagenesis, revealed residues 199-207 of the DSL domain to have a critical role in Notch binding (Cordle et al., 2008 [65], incorporated herein by reference). The same study mapped the predominant site of interaction on Notch 1 to the EGF12 domain.
US 2008/0317760 discloses a series of monoclonal antibodies raised in mice against residues 24-1060 of human Jagged 1, with epitopes mapping to the EGF1 domain. One antibody was identified as being capable of inhibiting Jagged 1-Notch1 association, and reducing tumour growth in a murine xenograft model. It is therefore apparent that, in the context of antibody-based therapy, targeting the DSL domain of human Jagged 1 is not essential for the inhibition of binding to endogenous receptors, such as Notch, and thus downregulated Jagged 1-mediated signalling.
WO 2011/063237 discloses further anti-Jagged 1 monoclonal antibodies, in this instance generated by the immunisation of mice with residues 1-1060 of mouse Jagged 1 and the identification of human Jagged 1 extracellular domain-recognising Fab fragments from a synthetic library using phage display. These were found to bind both human and mouse Jagged 1 with similar affinity, with all but one also recognising human Jagged 2. Several antibodies furthermore prevented human Jagged 1 binding to human Notch 2, inhibited Jagged-mediated signalling and reduced tumour growth in murine xenograft models. However, the epitopes recognised by the antibodies disclosed in WO 2011/063237 were not mapped to a distinct region or domain within the Jagged 1 protein.
In light of the prior art, there is a rationale for the provision of a therapeutic anti-Jagged 1 monoclonal antibody which recognises a defined epitope on human Jagged 1, moreover one that is distinct from the EGF domains, and which inhibits Jagged 1-mediated signalling and tumour growth with suitable efficacy for use in therapy. Given the well-documented occurrence of acquired resistance to cancer therapeutics due to single residue mutations, for example cases of imatinib-resistant chronic myeloid leukaemia [85], the provision of a therapeutic antibody with a defined epitope which is known to differ from that recognised by a pre-existing antibody against the same target, is of therapeutic value in the context of combination therapy. Furthermore, cross-reactivity of monoclonal antibodies between the EGF domains of different proteins has been previously reported, for example in the case of CD97 and EMR2 [86].
Accordingly, there is a need for additional antibodies capable of inhibiting the interaction between human Jagged 1 and Notch/CD46 through binding to a defined epitope on Jagged 1, preferably one which is distinct from the EGF domains.